The objective of this study is to construct an expression vector based on citrus leaf blotch virus (CLBV) and systematically express antimicrobial peptides to improve plant disease resistance, which will provide a new technical means for the prevention and control of citrus canker, citrus Huanglongbing (HLB) and other diseases.

The subgenomic promoter sequence and multiple cloning sites were inserted after the terminator of the coat protein gene of CLBV to construct the viral expression vector pCLBV202 based on the previously constructed infectious clone pCY-CLBV201. Then green fluorescent protein gene (gfp) was inserted into the multiple cloning sites, and the expression of GFP was verified by Agrobacterium-mediated inoculation and fluorescence observation. The cecropin B (CB) gene of Antheraea pernyi was cloned and the recombinant vector pCLBV202-CB was constructed. Nicotiana benthamiana and citrus seedlings were inoculated by pCLBV202-CB using Agrobacterium-mediated injection and vacuum infiltration, respectively. Positive plants were screened and subjected to inoculation of Ralstonia solanacearum by injection and root irrigation or Xanthomonas citri subsp. citri (Xcc) by pricking the detached leaves. At the same time, the plants inoculated with the empty vector were set as the control. The resistance to tobacco bacterial wilt of the inoculated plants was evaluated by symptom observation, incidence rate and disease index. The citrus resistance to canker was evaluated by the detriment, incidence rate and colony concentration on leaves.

After inoculated by pCLBV202-GFP, green fluorescence could be observed on the systemic leaves of the N. benthamiana and Citrus limon, which was brighter in N. benthamiana, indicating that the expression vector based on CLBV was successfully constructed. After inoculation of R. solanacearum, the onset time of treatment group (pCLBV202-CB) was delayed by 4 days compared with the control group (pCLBV202). On the 24th day past inoculation (24 dpi), the incidence rate of the treatment group was 14.3%, and that of the control group was 100%. Compared with the control group, the resistance index of the treatment group was -2.66, and the resistance evaluation was high, indicating that the expression of CB by pCLBV202-CB enhanced the resistance to tobacco bacterial wilt. When C. limon leaves was inoculated by Xcc, the number of detriment in the treatment group at 7 dpi was 47, and the incidence rate was 43.5%, while that of the control group was 73, and 67.6%. The detection of colony concentration changes of Xcc showed that the number of Xcc in the treatment group was less than that in the control group, indicating that the expression of CB by pCLBV202-CB enhanced the resistance to citrus canker.

The CLBV-based viral expression vector pCLBV202 was constructed. Using pCLBV202 to systematically express CB in N. benthamiana and citrus can improve the resistance of plants to bacterial diseases, which provided a new technology for the prevention and control of citrus bacterial diseases.